Lipid metabolism improving and anti-atheromatic agent

ABSTRACT

A lipid metabolism improving and anti-atheromatic agent comprises as an active ingredient a new lipolytic substance GA-56 capable of specifically decomposing chylomicron and low-density lipoprotein and having high intestinal absorbability.

United States Patent 1 Kobayashi 1 Apr. 1, 1975 1 LIPID METABOLISM IMPROVING AND ANTI-ATIIEROMATIC AGENT [75] Inventor: Mizuho Kobayashi. Gifu Japan [73] Assignee: Amano Pharmaceutical Co., Ltd., Nagoya, Japan [22] Filed: Feb. 6. 1973 [21] Appl. No.: 329,981

Related US. Application Data [63] Continuation-impart of Scr. No. 303.312. Nov. 3.

1972, abandoned [52] US. Cl 195/62, 195/65, 195/66 R [51] Int. Cl Cl2d 13/10 [58] Field of Search 195/62, 65, 66 R [56] References Cited UNITED STATES PATENTS 3.431.175 3/1969 Arima ct a1 195/62 3.511.753 5/1970 Pravc-ct a1. 195/62 OTl-IER PUBLICATIONS Chemical Abstracts. Vol. 72, 51163Z.

Lu et 211., Applied Microbiology. V01. 18. No. 1; pp. 104-111. July 1969-.

Primary Examinen-Lionel M. Shapiro Attorney. Agent. or FirmBrowdy and Neimark [5 7] ABSTRACT 6 Claims, 2 Drawing Figures LIPID METABOLISM IMPROVING AND ANTI-ATHEROMATIC AGENT CROSS-REFERENCE TO RELATED APPLICATIONS The present application is a continuation-in-part of US. application Ser. No. 303,312. filed Nov. 3. 1972. now abandoned.

BACKGROUND OF THE INVENTION It is known that the triglyceride taken as food is generally hydrolyzed in intestine and the hydrolyzate is synthesized again to triglyceride as chyromicron in the intestinal wall, from where it moves to the liver via blood and lymph vessels and is then resynthesized to the various lipoproteins. Therefore. various lipoproteins are present in blood while maintaining a suitable concentration relation in the state of normal lipid metabolism. However. once the lipid metabolism becomes abnormal. the various lipoprotein concentrations increase to cause hyper lipemia resulting in various diseases.

' stantially little inactivation in the presence of inorganic It has heretofore been believed that these lipopro- 1 teins are decomposed by lipoprotein lipases and that the resulting fatty acids are incorporated in, tissues. However, surprisingly, we have found that in rabbits fed with a high chlosterol diet for about six months there results fatty liver and hyper lipemia to high degree; and also the lipoprotein lipase activity was higher than in normal rabbits. Also in patients of hyper lipemia of Types II and IV according to .Fredericksons classification of hyper lipemia, it appears that the activity of the lipoprotein lipase is not different from the activity in normal subjects. Thus, the question hasexisted as to why disorders in lipid metabolism occur even in the absence of an interference with the activity of lipoprotein lipase.

BRIEF SUMMARY OF THE INVENTION Accordingly we considered that normal lipid metabolism, especially decomposition of lipoproteins, is not only influenced by the lipoprotein lipase released in blood, but that another factor dominates it, and we conducted research to determine what this other factor is. As a result, we have succeeded in confirming the presence of a lipase in liver which is different from the lipoprotein lipase present in plasma. fat tissue or heart muscle. And, it is considered that this lipase acts specifically on chylomicron in the liver toeffectively improve lipid metabolism. This hypothesis was demonstrated by the experiment showing that this lipase exhibits an abnormal activity in pathological animals suffering from abnormal lipid metabolism. When a disorder is brought about for some cause, hyper lipemia occurs, which results in more serious diseases such as hyper cholesteremia and fatty liver. In such case, sufficient improvement is not attained only by enhancing the activity of the lipoprotein lipase by administration of heparin or heparinoid. Recently these diseases could be treated only by the control of diet.

This invention relates to a lipid metabolism improving and anti-atheromatic agent comprising a new lipolytic substance having the following properties: 1) it acts specifically on natural fat and oil, chylomicron and low-density-lipoprotein to decompose them, (2) it hasan optimum activity at a pH range of from 7 to 1'1, (3

salts, (6) it is completely freeofsugar, (7) its molecular weight is approximately 30,000, and (8') its mobility as measured by paper electrophoresis is 1.53 X l0"" cm sec, V (pH 8.7, ;.t(ion strength) 0.05).

DETAILED DESCRIPTION OF THE INVENTION We believed that a new lipolytic substance having a lipase activity like the lipase in liver could be obtained from soil, especially microorganisms. so that when the new lipolytic substance was applied to the treatment of patients of hyper lipemia, the lipid metabolisms could be improved and normal metabolism could be recovered. Thus, we conducted extensive research on various microorganisms and succeeded in collecting a substance meeting the above requirements from bacteria. As a result of detailed examination of physical and chemical properties of this substance. it was confirmed that this substance is a new lipolytic substance which has heretofore been unknown. When this substance was administered to pathological animals suffering from hyper lipemia, it was found that the lipid concentration in blood could apparently be normalized with rapid improvement in lipemia or lipcmic plasma turbidity. We gave the name. new lipolytic substance GA- 56" to this substance. The new lipolytic substance (EA-56 will be referred to merely as GA-56 hereinbelow.

GA-56 which is an active ingredient of the lipid metabolism improving and anti-atheromatic agent of this invention is a new substance having physicochemical properties quite different from those of known lipases and lipoprotein lipases, Physicochemical properties of GA-56 will now be illustrated while clarifying differences of (EA-56 from known lipases and lipoprotein lipases..

1. Function:

GA-56 acts on ester linkages of triglyceride in various lipids and produces fatty acids and glycerine. 2. Substrate Specificity:

A variety of natural fats and oils are utilized as substrates for GA-56. GA-56 decomposes as substrates vegetable oils such as-olive oil, sesame oil, rape seed oil and peanut oil, animal oils such as lard and butter, fat and oils activated by albumin or serum protein, and lipoproteins, thereby to produce fatty acids and glycer- 3. Optimum pH range and stability pH range:

GA-56 acts within a broad pH range of more than 3, and an optimum pl-I: range is on the alkaline side, namely from 7 to II. When it is treated at 37C for 60 minutes, no inactivation is caused at a pH value of more than 4.

H Seiyaku) to provide a substrate solution. l.0 ml of the prepared substrate solution and 0.5 ml of 0.1 M phosphate buffer solution (:pH 7) are mixed in a test tube. and they are pre-incubated at 37C for 5 minutes.

Then, diluted GA- 56 is added to the tube and incubation is carried out at 37C for 20 minutes. Then, 5.0 ml

of stop solution is added and the mixture is shaken. followed by addition of ml of n-heptane and 2.0 ml of distilled water. .The mixture is shaken for 10 seconds. and is then. allowed to standstill. whereupon it separates into two phases. 3.0 ml of the heptane phase is pipettedby a safetygpipette. Indicator solution (1% of thymol blue in alcohol) is added dropvvise and nitrogen gas is blown in this solution and titrated with 0.01 N alcoholic potassium hydroxide solution by mieropipette. Titration is effected until a slight blue color appears. The blank value is obtained by employing distilled water instead of the diluted (EA-56 solution and conducting the .above procedures. The stop solution is a liquid mixture comprising 10 parts by volume of nheptane. parts by volume of isopropyl alcohol and 1 part by volume of 2 N sulfuric acid. The activity of releasing l micromole of fatty acid is defined as an activity of one unit.

5. Preferable Temperature for Function of GA-56:

The best function is observed at about 40 to C 6. lnactivating Temperature and pH Conditions:

Inactivation is extreme by the treatment at 37C for 60 minutes with an acidic liquor having a pH of less than 4. An aqueous solution of GA-56 is 70% inactivated by the treatment at 60C for 60 minutes.

7. Inhibition. Activation and Stabilization:

GA-56 is not at all inactivated by most inorganic salts. and the activity is increased in a sesame oil emulsion incorporated with albumin or serum protein.

8. Purification Method:

The crude powder of GA-56 obtained from culture broth is dissolved in water ofa volume 10 times as great as the powder. and the solution is centrifuged for 10 minutes at 10.000 r.p.m. The supernatant is salted out at a saturation of 0.2 with ammonium sulfate (grade No. l)and allowed to stand still for about 2 to 3 hours. Then, it is centrifuged again for 10 minutes at 10,000

9. Molecular Weight:

GA-56 has a molecular weight of about30,000 as measured by the gel filtration method. 10. Mobility:

(SA-56 has a mobility of 1.53 X l0 "cm'-, sec, V as measured by the paper electrophoresis (pH 8.7, ,u.

'l 1. Sugar Content:

GA-56 is negative at the sugar content test method. 12. Crystal Structure:

Since GA-56 is not obtained in the crystal form, purity of the substance was judged by electrophoresis (acrylamide disc electrophoresis method). As a result of the test conducted under conditions of 300V, 2 mA, 60 minutes and pH of 9.4. a single band wasobtained which corresponded to protein activity.

13. its ultra-violet absorption spectrum is as shown" in FIG. 2,

14. its infrared absorption spectrum by K Br disk is as shown in FIG. 1,

15. its elementary analysis gives C; 45.72%, H: 6.85%, O: 26.05%, N: 14.38%, S: 0.34%.

No substance having the same physicochemical properties as the above-mentioned properties of GA-56 has been found. Known substances resembling GA-56 are l an endogenous lipoprotein lipase observed in blood after intravenous injection of heparin and (2) a lipoprotein Iipase .microbiologically obtained (US. Pat. No. 3,431,175). GA-56 is substantially different from these known substances with respect to function, substratespecificity and the like. Further, it has been proved experimentally that GA- 56 is clearly. distinguished over the prior substances with respect to heat stability and that GA-56 is quite different from them vwith respect to influence by inhibitors. Differences between CIA-56 and a lipoprotein lipase obtained micro-" biologically disclosed in US. Patent No. 3,431,175 are. shown in Table 1 below.

TABLE I Lipoprotein Li ase disclosed in U Properties (IA-56 Pat. No. 3,431,175

microorganism used Iipolytic. activity influence by inhibitor sugar content electrophoretic mobility (cm sec. v)

r.p.m. Then, the precipitate is dissolved in water of a volume 10 times as great asthe precipitate. The solution is filtered with Sephadex G-50 gel washed preliminarily with distilled water. and active fractions are mixed together. The mixture is passed through a DEAE-cellulose column bufferized with 0.01 M phosphate buffer solution (pH 8). arid CIA-56 is absorbed thereon. The absorbed (SA-56 is eluted outby the linear gradient elution method using the above buffer solution contained with sodium chloride where the salt concentration is linearly heightened to 0.5 M. The active fraction is subjected again to the gel filtration with.

Sephadex G-75, followed by lyophilization toobtain a standard sample of GA'-56 exhibiting a single band corresponding to protein activity.

Pseudomonas Strain No. l56-A,ATCC No. 21,808 decomposition is observed when olive oil emulsion alone decomposition is not observed when olive 011 emulslon alone is present is present I not inhibited by inhibited with sodium sod um chloride, chloride, sodium 7 sociurn fluoride, fluoride, protamine protamine sulfate sulfate negative positive 1 tine have heretofore been reported by many investigators, but there is no establishedtheory of possibility of absorbed 'intes tinally only slightly. Anti-inflammatory enzymes such as trypsin and chymotrypsin and proteo- Iytic enzymes of other origins have been tested by the radioactive tracing method, the everted sac method,

intestinal absorption of such substances and the fact is that thereare some cases where such substances can be the intestinal perfusing method. etc. and the possibility of intestinal absorption of such substances is conjectured. However. test conditions have greatly deviated from the living state.

In contrast to these known substances. GA-65 of this invention has such a novel property that when solution containing GA-So dissolved in water at a certain concentration is injected directly in duodenum of rabbits and rats. the presence of the same substance as administered can be confirmed in peripheral blood.

More specifically. groups of rats (Wister strain; body weight 250 to 300 g; male) each group consisting of 5 rats. were fasted for 24 hours and (EA-56, obtained in Example 1 given hereinbelow. was injected into the duodenum of each rat in an amount of 6,000 u/kg. Blood was collected l hour after the administration, and serum was separated to determine the activity. The average value of 5 rats of one group was calculated. Results are shown in Table 11 below. In the group to which (IA-56 was administered, the activity was about times the value obtained in the control group. Thus. it can be confirmed that GA-56 can be absorbed directly from intestine.

As is seen from the results shown above. it has been confirmed that CIA-56 is very specifically absorbed from intestine of rats and the activity can be measured in blood. Further. in order to confirm the activity of GA-56 in blood. heat stability and influence by inhibitors of the serum after administration of (BA-56 in duodenum were examined.

Since GA-56 has a very high stability against heat, after administration ofGA-56 the serum was incubated in water bath at 56C for minutes. under which endogenous lipases. especially blood lipoprotein lipase, are completely inactivated. and the presence of the remaining activity was examined. As a result it was found that while an endogenous lipoprotein lipase was completely inactivated by the heating at 56C for 30 minutes. the serum collected after duodenum application still retains 76% activity after the same treatment. This value approximates 80% remaining activity obtained in the case of an aqueous solution containing 0.001% of GA-56. Thus it was confirmed that the activity of CIA-5o is quite different from that of the endogenous lipoprotein lipase with respect heat stability and that GA-56 can be absorbed from intestine.

lnfluences by sodium fluoride, protamine sulfate, sodium chloride and heparin have been reported as differences between lipoprotein lipases and other tissue lipases. Thus. we examined the effects of the above four substances on the serum after intraduodenal administration of .GA-56 and on the serum lipoprotein lipase obtained after intravenous injection of heparin. More specifically,, 1) (BA-56 was administered intraduodenally to a rabbit in an amount of 50,00 u/kg or (2) heparin was intravenously injected in a rabbit in an amount of ZO 'mg/Rg'. The above four inhibitors were allowed to act on each of the serum obtained in l and the serum obtained in (2) (lipoprotein lipase) at a concentration of0 to 1.0 M in the case of sodium chloride, 0 to 0.4 M in the case of sodium fluoride, and 0 to 2.0 mg/ml in the case of heparin and protamine sulfate; and the remaining activities were measured.

As a result, it was confirmed that serum l and serum (2) exhibited quite different effects against the inhibitors. More specifically. serum (2) was completely inactivated by sodium chloride at a concentration of 0.5 M and sodium fluoride at a concentration of0.2 M. while serum 1) exhibited remaining activities of and respectively. Further, serum (1) was not affected by protamine sulfate at 0.5 mg/ml whiIe the activity of serum (2) was found to be reduced to 80% by protamine sulfate at 0.5 mg/ml. Based on these experimental results. it was confirmed that GA-56 has properties quite different from those of endogenous lipoprotein lipases.

Such influences by inhibitors are generally observed in lipoprotein lipases. For instance. the microbial lipoprotein lipase disclosed in U.S. Pat. No. 3.431.175 has similar properties. It was thus confirmed that when GA-56 is duodenally administered, the presence of GA-56 in blood can be proved and that (SA-56 is a substance which is quite different from endogenous lipoprotein lipases with respect to influence against various inhibitors. In other words. it was confirmed that activities proved to be manifested in serum obtained after intraduodenal administration of GA-56 cannot be deemed to be due to endogenous lipoprotein lipase but they are exhibited by GA-56 absorbed from intestine.

Further, it was confirmed that GA-56, which can be absorbed from intestine as readily as mentioned above, has such a new specific substrate-decomposing property that, in vivo, experiments, GA-56 has a clearing of highly improving lipemia or lipemic plasma turbidity.

More specifically. rats (Wister strain; body weight to 200 g; male) after fasting for 24 hours were tested. A butter lipemia group of 5 rats were allowed to eat butter freely after fasting. and 3 hours later GA-56 was intramuscularly injected at a dose of 30,000 u/5 ml/kg. A Tween 80 lipemia group of 5 rats were intravenously injected with 400 ml/2 ml/kg of Tween 80 after fasting, and 3 hours later GA-56 was intramuscularly injected at a dose of 30,000 u/S ml/kg. One hour after injection of GA-56, blood was collected and the lipid concentration in serum was measured. Results are shown in Table 111 below in which each value is an average value of 5 rats.

TABLE III Tween 80 Administered Group As is seen from the results shown in Table III, in rats of the butter administered group and of the Tween 80 administered group, the triglyceride content in blood was reduced by 90% and 70%, respectively, by administration of GA-56. Thus. it was confirmed that GA-56 exhibits a prominent effect of improving lipemia and is obviously effective for decomposing and clearing lipemia composed mainly of dietetic chylomicron caused by butter administration and lipemia composed mainly of beta-lipoprotein caused by Tween 80 administration. In view of the chemical properties of GA-56, it may readily be conjectured that GA-56 ofthis invention can act greatly on chylomicron having a high lipid content and the like. It is also considered that it is a property peculiar to GA-56 in vivo that it has a high affinity with lipoprotein brought about by a surface active agent such as Tween 80 (condensation product of ethylene oxide and sorbitan mono-oleate; viscosity 600 to 800 cp at 25C.: density 1.05 to l.l).

From the above-mentioned experimental results it was confirmed that GA-56 administered into duodenum can be absorbed from the intestinal canal and manifests activities in blood to hydrize the lipoprotein and to clean lipid blood.

As is seen from the above experimental results, GA-56 of this invention is effective for improving hyper lipemia. and, since it can be absorbed from the intestinal canal, even when orally administered, GA-56 is suitable for long-term treatment by continuous administration. Further, it is possible to cure directly hyper lipemia (in blood) by intramuscular or intravenous injection of pure (EA-56.

Method of Manufacture of GA-56 Strains No. 156-A and No. l56-B were at first separated from soil as bacteria producing GA-56 of this invention.

Microbiological properties of these two strains will now be detailed. Strains No. 156-A and No. l56-B exhibit properties quite similar to each other and they differ only in very limited properties. Thus, only when the properties differ between these strains, description will be made individually. giving a special reference such as No. 156-A or No. l56-B. a. Morphology rods: (0.6 to 1.01.1.) X (2.0 to 3.0 1.) single or in pairs and in chains motile with single polar flagellum no spore gramstain: negative acid-fastners: negative b. Cultural Characteristics l. bouillon agar plate culture; circular growth of en-' tire convex, smooth surface of light grayish brown color, semi-transparent 2, bouillon agar slant culture; filiform growth with smoothentire, moderate degree of growth, circular, yellowish brown, slightly lustrous 3. bouillon liquid culture: moderate growth with thin pellicle on surface, slightly turbid 4. bouillon gelatin stab culture: moderate growth with liquefaction of infundi bule form 5. litmus milk: slightly alkaline. litmus-reductive, liquefaction with slight formation of precipitates c. Biological Properties 9 1. reduction of nitrate: positive (succinic acidsodium nitrate medium) 2. denitrification: negative 3. MR test: positive 4. VR test: negative 5. formation of indole: negative 6. formation of hydrogen sulfide: positive (weak) 7. hydrolysis of starch: negative 8. utilization of citric acid: negative 9. utilization of inorganic nitrogen source; nitrate is not utilized but ammonium salt is utilized l0. pigment formation:

i. klieglar medium: No. 156-A produces greenish yellow fluorescent pigment (water-soluble) but No. l56-B produces no pigment ii. king medium: red (slightly violet) pigment (No.

l56A), negative (No. l56-B) iii. tyrosin media: slightly brown pigment (watersoluble) iv. glutamic acid-agar medium: faintly yellow, watersoluble pigment ll. urease positive l2. oxidase: positive 13. catalase: positive 14. growing pH: 5 to 9 l5. growing temperature:

20C H-H 16. attitude to oxygen: aerobic l7. O-F test oxidative l8. formation of acid and gas from saccharides: i. formation of acid: as shown in Table [V below ii. formation of gas negative TABLE IV I I l The above taxological properties were determined according to -Manual of Microbiological Methods (compiled by the America] Society for Microbiology) and Summary of Microbiological Experiments" (compiled by Infectious Disease lnstitute, University of Tokyo). When these properties are considered in the system of the key of classification of bacteria in Bergeys Manual of Determinative Bacteriology", seventh edition, the above two strains are deemed to belong to the genus"Ps eudomonas," because they are rods with a single polar flagellum. have no function of forming spores and are characterized by having a positive catalase property, and being aerobic. When these properties are examined in detail relying on Journal of Fermentation". Vol. 49. No. 12. pages 968 to 980 1971), Medicinal Journal of Osaka University. Vol. 21. No. l. pages 1 to 6 1970) and The Journal ofGeneral Microbiology. Vol. 43. No. 2. it is considered that these strains resemble PXt'lIl/(IHIUHUS ji'ugi. Psemlonzomls ('tput'iu, and PAL'lll/UIHOHUA mepllirit'u. However, the above two strains are different from these known strains as shown in Table below.

TABLE V amount incorporated of such oil and fat is 0.5 to 1.09?

by weight based on the culture medium. The so formed culture media are infected with strains No. l56-A and Test Items Strain No.

Strain No. l56-B Gelatin liquefaction Litmus milk liqucfacliquefacliquefaction Pseudomonas Pscudomonas Pscudomonas mephitica lragi ccpacia solidification I As is seen from the foregoing. taxilogical properties of strains No. 156-A and No. 156-8 to be utilized in this invention do not coincide with those of any known strain belonging to the genus Pseudomonas. Further, each of these strains with respect to microbiological properties are very similar. Therefore, it is deemed that these two strains may be included in one species. Accordingly, we have established novel species Pseudomonus nor. sp. No. 156 and both strain No. 156-A and strain No. 156-8 have been classified as belonging to the above new species.

In other words, Pseudo/norms not. sp. No. 156 has strain No. 156-A and strain No. 156-8 as standard strains and includes varieties and mutants thereof.

Both strains No. l56-A and No. 156-8 were deposited with the Fermentation Research Institute, Agency of Industrial Science and Technology, Japan, and they are preserved as FERM-P No. 1431 and FERM-P No. 1432, respectively, at said depository. Furthermore, strains No. 156-A and No. l56-B were deposited with the America] Type Culture Collection, Rockville, Md., and deposition numbers ATCC 21,808 and ATCC 21,809 have been allotted to strains No. 156-A and No. l56-B respectively. All restrictions on the availability to the public of these strains will be irrevocably removed upon the granting of the patent and the deposit will be maintained to assure the permanent availability thereof to the public throughout the effective life of the patent resulting from this application.

Ordinary liquid culture and solid culture methods may be utilized for culture of strains No. 156-A and No. 156-8. It is preferred that these strains be cultured in a culture medium which incorporates oil and fat into a fundamental medium comprising glucose, cornsteep liquor. urea, monopotassium hydrogen phosphate, po-- tassium chloride and magnesium sulfate.

As the oil and fat to be incorporated in the above fundamental culture medium, there may be mentioned, for instance, vegetable oils such as olive oil, sesame oil, rape seed oil and peanut oil. soybean oil, rice oil, cocnut oil. corn oil, castor oil, lineseed oil, cacao fat, etc., and animal oils such as lard, butter, beef tallow, sheep fat, etc. These oils and fats allow sufficient propagation of the above strains and they play an important role as carbon source for producing (IA-56. In general, the

No. l56-B, and culturing is effected aerobically at 25 to 28C, In about 24 to 30 hours, production of CIA-56 reaches a maximum. Isolation of GA-56 from the resulting culture broth can be accomplished by removing the cells from the culture broth by a centrifugal separator. concentrating the filtrate in vacuo, adding to the concentrate cold ethyl alcohol or acetone in a volume 1 to 3 times the volume of the supernatant, and removing the precipitate by centrifugation, adding to the supernatant ethyl alcohol or acetone of a volume 2 to 3 times the volume of the supernatant, washing the resulting precipitates with ethyl alcohol or acetone and drying them in vacuo at room temperature. Instead of the above-mentioned organic solvent precipitation method, it is possible to adopt, for instance, a method comprising salting out the clear filtrate with ammonium sulfate or other salt, subjecting the resulting precipitate to dialysis so as to remove the salt therefrom, and then lyophylizing.

Formulations of GA-56 (EA-56 may be formed into drugs such as tablets, capsules, powders, injections and suppositories. In the case of drugs for oral administration, it is preferred that GA-56 drugs are so formed that they become soluble after they reach the intestines. In the case of injection formulations, GA-56 is used in the highly purified form. In the case of oral drugs or suppositories, GA-56 may be used in the form of crude powder or the roughly purified state, and it is incorporated with an extender, a binder and a suitable base such as cocoa butter and the admixture is molded into tablets, filled in capsules or formed in suppositories. Exterior coatings of known compositions may be used for tablets and capsules for oral administration.

This invention will .now be illustrated in more detail by references to Examples which are offered illustratively.

EXAMPLE 1 a 30-liter capacity and sterilized at 120C for 30 minutes in high pressure. Then. the culture broth was infected with 600 ml of a pre-cultured broth obtained by pre-culturing Pxcmlrmionus I101. .vp. No. 156-A strain at 26C for 24 hours in a similar culture broth as that defined above. The culture was effected at 26C. 200 r.p.m. and 0.4 vvm under agitation and'passing air through the culture broth. In 24 hours. the production of GA-56 reached a maximum.

After completion of the culturing. the culture broth was centrifuged by means of a centrifugal separator to remove the cells. and the filtrate was concentrated in vacuo at 30C until the volume was reduced to onefiftli. Cold ethyl alcohol of a volume 2 times the volume of the concentrate was gradually added to the concentrate under agitation. and the resulting precipitates were filtered by means of a filter press using diatomaceous earth as a filtering assistant. Then. the filtrate was combined under agitation with cold ethyl alcohol of a volume 2.5 times the volume of the filtrate to form precipitates again. The supernatant liquor was removed. and the remaining precipitates were washed sufficiently with cold ethyl alcohol. centrifuged to remove ethyl alcohol and dried in vacuo at room temperature to give 33 g of white powder of GA-56 having an activity of 58.000 u/g.

EXAMPLE 2 Fifteen liters ofa liquid culture broth (pH 6) containing 3% of cornsteep liquor. 1% of glucose. 0.6% of urea. 0.2% of dipotassium hydrogen phosphate. 0.05% of potassium chloride. 0.05% of magnesium sulfate and 1.5% of olive oil was poured into a jar fermenter of a 30-liter capacity, and sterilized at 120C for 30 minutes at high pressure. Then. the culture broth was inplanted with 600 ml of a pre-cultured broth'obtained by preculturing Psemlomonas nuv. sp. No. l56-B strain for 24 hours in a culture broth similar to that described above. The culture was effected at 26C. 200 rpm, and l vvm in an agitated state by passing the air through the culture broth. In 28 hours, the production of GA-56 reached a maximum. After completion of the culture, post-treatments were carried out in the same manner as in Example 1 to obtain 25g of white powder of GA-56 having an activity of 52.000 u/g.

1 EXAMPLE 3 Fifteen liters of a liquid culture broth (pH 6) containing 0.6% of urea, 0.2% of dipotassium hydrogen phosphate. 0.05% of potassium chloride. 0.05% of magnesium sulfate anad 1.0% of lard was poured into ajar fermenter of a 30-liter capacity. and after sterilization at 120C for 30 minutes at high pressure it was infected with600 ml of a pre-cultured broth obtained by pre-culturing Pseudo/norms not. sp. No. 156-A strain for 24 hours in a culture broth similar to that described above. The culture was effected at 26C, 200 r.p.m. and 0.5 vvm in an agitated state by passing the air through the culture broth. In 30 hours. the production of GA-56 reached a maximum. In the same manner as in Example 1, post-treatments were carried out to obtain 40g of white powder of GA-56 having an activity of 54.000 u/g.

EXAMPLE 4 I I Sugar Coated Tablets (Enteric Coated Tablets) A. Composition (one tablet of 100 mg):

( l-l (IA-56 10,000 a (25%) (2) synthetic aluminum silicate crystalline cellulose 3% lactose 3% (3) calcium carhoxymethyl cellulose 5 (4) Hydroxy Propyl Cellulose-SI. 1%

(5) magnesium stcarate 0.5% to) parts of hydroxypropylmethyl I cellulose phthalate and 1 part of diethyl phthalate 5% 7) sugar coating 50% (N) tar pigments trace,

B. Preparation Method:-

Ingredients l to (5) were blended in amounts indicated above. and strip tablets were prepared from the blend according to the tablet method specified in J.P. VIII Pharmaceutical General Rule. An enteric coated film was applied to the so-formed strip tablets with use of the ingredient (6) in an amount indicated above according to the film coating method specified in J.P. VIII Pharmaceutical General Rule. Then, a sugar coating was applied to the so filmcoated tablets with use of ingredients (7) and (8) in amounts indicated above according to the sugar coating method specified in J.P. VIII Pharmaceutical General Rule.

C. Method of administration:

One tablet 3 times daily (.600 units per Kg of body weight per day), after meals. II Drugs for Internal Use (Enteric Coated Capsules) A. Composition (one capsule of 200 mg):

GA-56 powder 25.000 p.(50%) synthetic aluminum silicate crystalline cellulose 10% lactose 10% B. Preparation Method GA-56 25.000 p. (2) one part of methyl paraben and one part of butyl paraben cacao or suitable suppository base trace suitable amount B. Preparation Method From the above ingredients were prepared suppositories according to the suppository method specified in J.P. VIII Pharmaceutical General Rule.

C. Methodofadministration One suppository daily (500 units per'Kg of body weight per day).

Administration Test (Enteric Coated Tablets) Rabbits (body weight 2.5 kg; male) were intravenously injected with 200 mg/kgof Triton WR 1339 to form a lipemia. Twenty-two hours later, enteric coated tablets were forcibly administered orally to the rabbits at a dose of 10.000 u/kg of body weight.

Five ml of blood was collected from ear vein at every prescribed period. and serum was separated therefrom to determine the triglyceride content in serum and the activity of GA-56. Results are shown in Table VI below. As is seen from these results. in the control group even after 6 hours. the content of the endogenous lipid in blood. i.e.. triglyceride. was as high as 94% of the triglyceride content observed 22 hours after injection of Triton WR 1339. On the other hand. in the GA-56 administered group. in 2 hours. the lipid content was already reduced. and in 6 hours. the lipid content was reduced by about 35% This coincides with the fact that in the control group hardly any change was observed in the activity in blood. while in the GA-56 administered group. in 4 to 6 hours. the activity was almost tripled.

ln view of the foregoing. it can be judged that GA-56 Table V1 given together with the cholesterol diet. 1n another group (group 111). after the cholesterol feeding was continued for 8 weeks. administration of the drug ob tained in this Example was started. In the control group (group 1). the cholesterol feeding was effected without administration of the drug obtained in this Example. In this manner. the cholesterol diet feeding was continued for 22 weeks in each group. The administration of the drug (capsule) was effected once every day forcibly orally at a dose of 10.000 u/kg as calculated as active ingredient GA-56.

In each group. the abdominal incision was conducted under general anesthesia. and aorta arcus was removed. This tissue was fixed with formalin. and frozen sections having a thickness of 15a were sliced therefrom. Then. after staining with Sudan-1V these preparates were examined hystochemically by high power microscope.

Triglyceride Content (mg/d1) Activity (p/ml) Time GA-56 administered GA-56 administered (hours) control group group control group group 30 As a result. it was found that in the rabbits of group has the ability to decompose prominently endogenous lipids and is very effective for improvement of the lipid metabolism.

EXAMPLE Drugs for Internal Use (Capsules with Enteric Coated Granules) A. Composition (one capsule of 400 mg):

10.000 u 2) synthetic aluminum silicate 32% B. Preparation Method: 1

Ingredients (1) to (4) were blended in amounts indicated above, and the blend was molded into globular granules (15 to 50 mesh) according to the granule method specified in J. P. V111 Pharmaceutical General Rule. Then. the granules were coated with an enteric film with use of ingredients (5) and (6) of amounts indicated above according to the film coating method specified in J. P. V111 Pharmaceutical General Rule. Then. prescribed amounts of the enteric film-coated granules were filled up into capsules. C. Method of administration:

One capsule 3 times daily (600 units per Kg of body weight). after meals.

Administration Test Three groups of rabbits (body weight 2.5 kg;

male). each group consisting of5 rabbits, were fed with v 1. the obvious atherosclerotic picture was observed in both of the inner layer and the middle layer. In 40% of the rabbits of group 11. no lipid deposition was observed and in the remaining 60%. only slight lipid deposition was observed in the inner layer. In each of the rabbits of group 111. lipid deposition was observed in the inner layer. and when the degree of the deposition was compared by extraction of the lipid, it was found that the degree of the lipid deposition in Group 111 was only 20 to 30% of the deposition degree in the control group 1. It was also found that in the tissue preparation obtained from the rabbits administered with the capsule of GA-56 there was formed a tissue picture deemed to be due to the effect of GA-56 in amelioration against the formation of atheromatics.

EXAMPLE 6 Drug for Internal Use (Injection) A. Composition (one vial):

(l) purified GA-56 50 p. (2) dissolving liquor (isotonic sodium chloride solution) 2 ml Purified GA-56 was dissolved in the dissolving liquor when injection was effected. B. Preparation Method The purified GA-56 (exhibiting a single band at the disc electrophoresis) was aseptically filled in a prescribed amount according to JP V111 Pharmaceutical General Rule to obtain a lyophilized ampule. The dissolving liquor was prepared by dissolving sodium chloride in injectional sterile distilled water. filtering the solution. filling the filtrate into ampules and effecting the sterilization at 115C for 30 minutes. 1 C. Method of administration 1 One vial daily (1 unit per Kg of body weight per day).

Administration Test (10) its infrared absorption spectrum is as shown in Triton WR 1339 was injected intravenously in an FIG. 1, and (11 its elementary analysis gives C:

amount of200 mg/kg of body weight into rabbits (body 45.72%, H: 6.85%, O: 26.05%. N: 14.38%, S: 0.34%. weight 2.5 kg male) to form a lipemia. After 22 2. A process for the production of lipolytic substance hours. one ampule of the purified GA-56 sample was GA-56 which comprises dissolved in 2 ml of sterilized physiological saline and culturing bacteria selected from the group consisting intravenous injection was conducted at a dose of 10 of Pseudomonas l56-A ATCC No. 21,808 and u/kg of body weight. v Pseudomonas 156-B ATCC No. 21,809 in an aque- Five ml of blood was collected from an ear vein at m ous nutrition medium containing an assimilable every prescribed period, and serum was separated to carbon source. an assimilable nitrogen source and determine the triglyceride concentration in serum and an inorganic nutrient source'under aerobic condithe activity. Asa result. it was found that the triglycertions at to 40C until lipolytic substance GA-56 ide concentration in blood tended to increase in 4 is accumulated in the culture broth.

hours in the control group. whereas reduction of about H 3. A process for the production oflipolytic substance in the triglyceride concentration was observed in GA-56 which comprises cultivating bacteria selected the GA-56 administered group. Thus, as in Example 4 from the group consisting of Pseudomonas strain No. it was confirmed that CIA-56 is effective for improving l56-A ATCC No. 21,808 and Pseudomonas strain No. the lipid metabolism. Results obtained in' this Example l56-B ATCC No. 21,809 in an aqueous nutrition meare shown in Table VI) below. dium containing an assimilable carbon source, an inor- TABLE vii Triglyceride Concentration (mg/d1) Activity in Blood a/ml) Time CIA-56 administered GA-56 administered (hours) control group group control group group Having thus described my invention the following ganic nutrient source and a member selected from the claims set forth that which is to be secured by letters group consisting offat and oil under aerobic conditions patent of the United States. at 20 to 40C for 20 to 40 hours until lipolytic sub- 1 I stance GA-56 is accumulated in the culture broth, and C isolating lipolytic substance GA-56 from the obtained 1. Lipolytic substance GA-56 capable of specifically culture. hydrolyzing chylomicron and low-density lipoprotein 4. A process according to claim 3 wherein said memand having high intestinal absorbability, said lipolytic ber selected from the group consisting of fat and oil is substance (EA-56 having the following properties (1) it added to the medium in the range of 0.5 to 2.0% by acts specifically on natural fat and oil, chylomicron and weight.

low-density lipoprotein to decompose them, (2) opti- 5. A process according to claim 3 wherein said memmum pH range is from 7 to 1 l, (3) optimum temperaber selected from the group consisting of fat and oil is ture is between 40 and 50C, (4) it suffers no inactivaolive oil, sesame oil, rape seed oil, peanut oil, soybean tion in 60-minute treatment at 37C and at pH 4, (5) oil, rice oil, castor oil, linseed oil, corn oil, coconut oil, it suffers substantially little inactivation in the presence cacao fat, lard, butter, beef tallow or sheep fat.

of inorganic salts, (6) it is completely free of sugar, (7) 6. A process according to claim 4 wherein said memits molecular weight is approximately 30,000, (8) its ber selected from the group consisting of fat and oil is mobility as measured by paper electrophoresis is olive oil, sesame oil, rape seed oil, peanut oil, soybean 153x [0 cm", sec, v (pH=8.7, p,=0.05), (9) its uloil, rice oil, castor oil, linseed oil, corn oil, coconut oil, tra-violet absorption spectrum is as shown in FIG. 2, cacao fat, lard, butter, beef tallow or sheep fat.

UNITED STATES PATENT OFFICE CERTIFICATE GF CORRECTION PATENT NO. 3,375 007 DATED April 1, 1975 mv ENTO R( s) Mizuho KOBAYASHI It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:

Column Column Column Column [SEAL] 1, line 32, "II and IV" should read -II to IV- 11, line 53, "anad" should read --and-- 12, line 48, delete "8n" at the end of the line Signed and Scaled this twenty-ninth Day of July 1975 A ttes t:

RUTH C. MASON Arresting Officer C. MARSHALL DANN ('rmmzissl'mzcr uflurenls and Trademarks 

1. LIPOLYTIC SUBSTANCE GA-56 CAPABLE OF SPECIFICALLY HYDROLYZING CHYLOMICRON AND LOW-DENSITY LIPOPROTEIN AND HAVING HIGH INTESTINAL ABSORBABILITY, SAID LIPOLYTIC SUBSTANCE GA-56 HAVING THE FOLLOWING PROPERTIES (1) IT ACTS SPECIFICALLY ON NATURAL FAT AND OIL, CHYLOMICRON AND LOW-DENSITY LIPOPROTEIN TO DECOMPOSE THEM, (2) OPTIMUM PH RANGE IS FROM 7 TO 11, (3) OPTIMUM TEMPERATURE IS BETWEEN 40* AND 50*C, (4) IT SUFFERS NO INACTIVATION IN 60-MINUTE TREATMENT AT 37*C AND AT PH 4, 0008 (5) IT SUFFERS SUBSTANTIALLY LITTLE INACTIVATION IN THE PRESENCE INORGANIC SALTS, (6) IT IS COMPLETELY FREE OF SUGAR, (7) ITS MOLECULAR WEIGHT IS APPROXIMATELY 30,000, (8) ITS MOBILITY AS MEASURED BY PAPER ELECTROPHORESIS IS 153$10-5 CM2, SEC-1, V-1 (PH=8.7, U=0.05), (9) ITS ULTRA-VIOLET ABSORPTION SPECTRUM IS AS SHOWN IN FIG. 2, (10) ITS INFRARED ABSORPTION SPECTRUN IS AS SHOWN IN FIG. 1, AND (11) ITS ELECENTARY ANALYSIS GIVES C: 45.72%, H: 6.85%, 0:26.05%, N: 14.38%, S: 0.34%.
 2. A process for the production of lipolytic substance GA-56 which comprises culturing bacteria selected from the group consisting of Pseudomonas 156-A ATCC No. 21,808 and Pseudomonas 156-B ATCC No. 21,809 in an aqueous nutrition medium containing an assimilable carbon source, an assimilable nitrogen source and an inorganic nutrient source under aerobic conditions at 20* to 40*C until lipolytic substance GA-56 is accumulated in the culture broth.
 3. A process for the production of lipolytic substance GA-56 which comprises cultivating bacteria selected from the group consisting of Pseudomonas strain No. 156-A ATCC No. 21,808 and Pseudomonas strain No. 156-B ATCC No. 21,809 in an aqueous nutrition medium containing an assimilable carbon source, an inorganic nutrient source and a member selected from the group consisting of fat and oil under aerobic conditions at 20* to 40*C for 20 to 40 hours until lipolytic substance GA-56 is accumulated in the culture broth, and isolating lipolytic substance GA-56 from the obtained culture.
 4. A process according to claim 3 wherein said member selected from the group consisting of fat and oil is added to the medium in the range of 0.5 to 2.0% by weight.
 5. A process according to claim 3 wherein said member selected from the group consisting of fat and oil is olive oil, sesame oil, rape seed oil, peanut oil, soybean oil, rice oil, castor oil, linseed oil, corn oil, coconut oil, cacao fat, lard, butter, beef tallow or sheep fat.
 6. A process according to claim 4 wherein said member selected from the group consisting of fat and oil is olive oil, sesame oil, rape seed oil, peanut oil, soybean oil, rice oil, castor oil, linseed oil, corn oil, coconut oil, cacao fat, lard, butter, beef tallow or sheep fat. 